Sutapa Sarkar
1, Probir K. Bandyopadhyay
1, Bayram Göçmen
21Department of Zoology, University of Kalyani, West Bengal, India
2Department of Biology, Faculty of Science, Ege University, İzmir, Turkey
ABSTRACT
Objective: Biodiversity studies in search of endoparasitic acephaline gregarines of earthworms revealed a new species under the genus Rhynchocystis Hesse, 1909.
Methods: The species has been obtained from the seminal vesicles of the earthworm, Metaphire peguana. The parasite was identified us- ing standard methodology.
Results: Gamonts are solitary, elongated with a conical, enlarged head and blunt posterior end. The mature gamont measures 419.90- 430.95 (425.05±4.70) µm in length and the width at the widest portion is 44.2-50.83 (47.88±2.85) µm and 15.47-17.68 (16.50±1.14) µm in the posterior end. The nucleus is elongated and positioned at the posterior end. The nucleus measures 17.68-22.01 (19.74±1.95) µm×11.05- 15.47 (13.40±1.95) µm. The measurement of the mucron is 13.26-17.68 (15.47±1.86) µm×17.68-22.1 (19.44±1.71) µm. Gametocysts are ovoid with two unequal sized gametocytes. The gametocyst measures 95.03-97.24 (96.20±1.14) µm×77.35-81.77 (79.56±1.86) µm. Large and small gametocytes measure 57.46-61.88 (59.81±1.76) µm×70.72-75.14 (72.48±1.71) µm and 30.94-37.57 (34.47±2.74) µm×61.88-66.3 (64.38±1.84) µm respectively. Oocysts are biconical, measuring 11.05-15.47 (12.81±1.71) µm×6.63-8.84 (7.70±1.20) µm.
Conclusion: A new gregarine parasite species is described. (Turkiye Parazitol Derg 2012; 36: 178-81) Key Words: Rhynchocystis silvae sp. nov, gregarine, earthworm, seminal vesicles, Bangladesh Received: 24.04.2012 Accepted: 09.06.2012
ÖZET
Amaç: Toprak solucanlarının endoparazitik asefalin gregarinlerinin araştırılmasına yönelik biyoçeşitlilik çalışmaları Rhynchocystis, Hesse, 1909 cinsine dahil yeni bir türü ortaya koymuştur.
Yöntemler: Yeni saptanan tür, Metaphire peguana’nın seminal vesiküllerinden elde edilmiştir. Parazitin teşhisinde standart yöntemler izlen- miştir.
Bulgular: Gamontlar soliter, konik şekilde uzamış bir baş ve küt bir posterior uca sahiptir. Ergin gamont uzunluğu 419.90-430.95 (425.05±4.70) µm ve genişliği posterior uçta 15.47-17.68 (16.50±1.14) µm iken en geniş kısımda 44.2-50.83 (47.88±2.85) µm olarak ölçülmüştür. Nukleus uzamış olup, posteriorda yerleşir. Nukleus ölçüleri 17.68-22.01 (19.74±1.95) µm×11.05-15.47 (13.40±1.95) µm’dir. Mukron ölçüleri 13.26- 17.68 (15.47±1.86) µm×17.68-22.1 (19.44±1.71) µm’dir. Gametosistler oval ve farklı büyüklükte iki gametosit içerir. Gametositler 95.03-97.24 (96.20±1.14) µm×77.35-81.77 (79.56±1.86) µm’dir. Büyük ve küçük gametosit ölçüleri sırasıyla, 57.46-61.88 (59.81±1.76) µm×70.72-75.14 (72.48±1.71) µm ve 30.94-37.57 (34.47±2.74) µm×61.88-66.3 (64.38±1.84) µm’dir. Ookistler bikonik ve 11.05-15.47 (12.81±1.71) µm×6.63-8.84 (7.70±1.20) µm boyutlarındadır.
Sonuç: Yeni bir gregarin parazit türü tanımlanmıştır. (Turkiye Parazitol Derg 2012; 36: 178-81)
Anahtar Sözcükler: Rhynchocystis silvae sp. nov., gregarin, toprak solucanı, seminal vesikül, Bangladeş Geliş Tarihi: 24.04.2012 Kabul Tarihi: 09.06.2012
Address for Correspondence / Yazışma Adresi: Dr. Bayram Göçmen, Department of Biology, Faculty of Science, Ege University, İzmir, Turkey Phone: +90 232 388 40 00 Fax: +90 232 388 10 36 E-mail: bayram.gocmen@ege.edu.tr
doi:10.5152/tpd.2012.42
Description of Rhynchocystis silvae sp. nov (Apicomplexa:
Eugregarinida) From Metaphire peguana Rosa (1890) of Dhaka, Bangladesh
Dakka, Bangladeş’den Bir Solucan Türü Metaphire peguana Rosa (1890)’da Yeni Bir Gregarin Parazit Türü Rhynchocystis silvae sp. nov. (Apicomplexa: Eugregarinida)
178
Original Investigation / Özgün AraştırmaINTRODUCTION
“Gregarines” are a diverse and successful group of protozoan parasites belonging to the phylum Apicomplexa. Gregarines are specific parasites of a wide range of invertebrates from Phyla Arthropoda (including crustaceans, insects, myriapods) and Annelida (including polychaetes, oligochaetes and leeches). Of the two major groups of gregarines, aseptate and septate, earth- worms harbour the aseptate one. The aseptate or acephaline forms are characterized by a non-septate body and they are the common endoparasites of the seminal vesicles of earthworms.
There are not many important studies on gregarine protozoan parasites infesting invertebrates throughout the world. The researches on aseptate gregarines have gained momentum since 1980. Ruston (1) described D. minuta from Lumbricus terrestris.
Bandyopadhyay and Mitra (2) described one species, D. indica from Lampito mauritii collected from India. Later, a study of asep- tate gregarines from earthworms revealed a new species of the genus Rhynchocystis Hesse 1909, which is described here. Levine (3) listed some species under the genus Rhynchocystis. These were Rhynchocystis awatii, R. cognetti, R. cuneiformis, R. pilosa, R.
hessei, R. mamillata, R. oculata, R. ovata, R. pessoai, R. piriformes, R. porrecta. All of these species of Rhynchocystis were described from seminal vesicles of oligochaete hosts. During the present biodiversity studies of the aseptate gregarines from the earth- worms of Bangladesh, a new species belonging to the genus Rhynchocystis previously defined by Hesse in 1909 was identified.
This paper deals with a unique form of a Rhynchocystis species obtained from the seminal vesicles of the earthworm, Metaphire peguana, collected from North Badda of the Dhaka district, Bangladesh. This new species of aseptate gregarine is described for the first time in the present study.
METHODS
For the present study 30 host specimens were collected from the drainage soil of North Badda, Dhaka. The specimen collections were conducted between January 2011-June 2011. The collect- ed earthworms were kept in soil in an earthen tub and brought to the laboratory alive. Some of the earthworms were dissected while alive and their seminal vesicles were carefully removed.
The fluids of the seminal vesicle were placed on clean glass with a drop of 0.6% NaCl solution. A thin film of seminal fluid was drawn out on a slide and covered with a cover slip for examina- tion of living protozoans under a light microscope. After initial study of living protozoans the content of the seminal vesicles were semidried and fixed in Schaudin’s solution for 20 minutes.
After fixation, the smears were stored in 70% ethanol for remov- al of mercuric chloride. The slides were then passed through a descending series of alcohol (5 minutes each) and placed in distilled water. These were transferred to a 3% iron alum solution (Over night) and stained with Heidenhain’s hematoxylin solution for 20 minutes. Differentiation was done with 1% iron alum solu- tion under the low power objective lens of the light microscope.
The slides were then washed thoroughly, dehydrated in an ascending series of alcohol, cleared in xylin and mounted in D.P.X. All measurements are presented in micrometers (µm) as mean (±) SD followed in parentheses by the range. Photographs were taken with an Olympus Phase Contrast Microscope fitted with a digital camera. The methodology used to describe shapes of planes and solids of the gregarines is in accordance with
Clopton (4). The following abbreviations are used for the grega- rine parasite in this paper. GL=Gamont length, GWwa=Gamont width at the widest part (anterior end), GWp=Gamont width (posterior part), LN=Length of nucleus, WN=Width of nucleus, LG=Length of gametocyst, WG=Width of gametocyst, LGL=Large Gametocyte Length, LGW=Large Gametocyte Width, SGL=Small Gametocyte Length, SGW=Small Gametocyte Width, LO=Length of oocyst, WO=Width of oocyst.
RESULTS
Phylum-Apicomplexa Levine, 1988 Order - Eugregarinida Leger, 1900 Family- Monocystidae Biitschli, 1882 Subfamily - Rhynchocystinae Bhatia 1930 Genus- Rhynchocystis, Hesse, 1909
Rhynchocystis silvae sp. nov (Figure 1 a-c, Table 1, 2) Mature gamont measures 419.90-430.95 (425.05±4.70) µm×15.47- 17.68 (16.50±1.14) µm. The widest part of it is 44.2-50.83 (47.88±2.85) µm width. The nucleus measures 7.68-22.01 (19.74±1.95) µm×11.05-15.47 (13.40±1.95) µm. Mucorn measures 13.26-17.68 (15.47±1.86) µm×17.68-22.1 (19.44±1.71) µm. The gametocyst is 95.03-97.24 (96.20±1.14) µm in length and 77.35- 81.77 (79.56±1.86) µm in width. Large and small gametocytes measure 57.46-61.88 (59.81±1.76) µm×70.72-75.14 (72.48±1.71) µm and 30.94-37.57 (34.47±2.74) µm×61.88-66.3 (64.38±1.84) µm respectively. Length of the oocyst is 11.05-15.47 (12.81±1.71) µm and width is 6.63-8.84 (7.70±1.20) µm.
The genus Rhynchocystis was detected by Hesse in 1909. Bhatia, placed it in a new sub family Rhynchocystinae in 1930 and the subfamily was defined as, “Gamont ovoid, spherical or elongated with a conical or cylindroconical trunk at the anterior end, solitary:
oocyst biconical with similar non appendiculate ends, with eight sporozoites” by Levine (1). The morphology of the present form obtained from the seminal vesicles of the earthworm, Metaphire peguana collected from Dhaka, the capital of Bangladesh is stud- ied thoroughly. The trophozoite of the present form is usually long
Figure 1. a-c) Photomicrographs of different stages of the life cycle of Rhynchocystis silvae sp. nov. obtained from the seminal vesicles of the earthworm, Metaphire peguana. a) A mature gamont; b) Gametocyst with two unequal sized gametocytes; c) Oocysts
a
c b
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and slender. Its anterior end is broad and rounded but the rest of the body tapers towards the posterior extremity. It is cylindrical and blunt. The anterior end of the parasite is provided with a small conical mucorn which emerges from the base of a concavity formed by the infolding of the ectosarc. Endoplasm is dense with paraglycogen grains. Epicyte is thin, transparent and quite deli- cate. Ectoplasm is relatively thick. The nucleus is small, elongated and near the posterior end, typically contains a central karyosome surrounded by a layer of chromatic granules which lies half way between it and the thick nuclear membrane. Gametocysts have two unequal sized gametocytes. Oocysts are biconical. Detailed measurements are presented in Table 1.
Taxonomic Summary
Type Material: Rhynchocystis silvae sp. nov Type Host: Metaphire peguana.
Type Locality: North Badda, (23.7667° N and 90.4333° E.) Dhaka district, Bangladesh
Site of infestation: Seminal Vesicles.
Prevalence: 21/30=(70%)
Holotype: R1/301/2011, deposited at the Parasitology Laboratory of the Department of Zoology, University of Kalyani, Kalyani -741235, West Bengal, India.
Paratypes: R2/302/2011, deposited at the Parasitology Laboratory of the Department of Zoology, University of Kalyani.
Kalyani-741235, West Bengal, India.
Symbiotype: MP/11/09, deposited at the Parasitology Laboratory of the Department of Zoology, University of Kalyani.
Etymology: The species is named in honour of Dr. Joseph D’Silva, former Professor of the Department of Zoology from the University of Dhaka, Bangladesh for his contribution in the field of Parasitology.
DISCUSSION
Being a parasite of seminal vesicles of oligochaetes and having a cylindrical anterior end, with little differentiation, the parasite justifies its inclusions under the taxa: family Monocystidae, Sub- family Rhynchocystinae and the genus Rhynchocystis Hesse, 1909. The genus Rhynchocystis is characterized by the presence of some sort of anterior epimerite like apparatus. Only ten Rhynchocystis species have been described so far from all over the world by Levine (1), and only few of them are described from India. The species presented here has been described from the earthworm of Bangladesh for the first time. Among the described species, R. pilosa and R. porrecta are more closely related with the new gregarine species presented in this study by Troisi (5). In R. pilosa the epimerite like apparatus is highly specialized for attaching the parasite with host cells, whereas the other species of the same genus rarely or never uses its anterior apparatus for the purpose of attachment and it may be reduced to a small, conical, protoplasmic structure called the mucorn by Troisi, (5). In R. pilosa the body is usually long and slender, anterior end broad and rounded, while the rest of the body tapers toward the pos- terior extremity, which is pointed. The average size of R. pilosa is 217.3µ×25.5µ. The present form differs from R. pilosa in having a more elongated and cylindrical body with a blunt posterior end and it measures 419.90-430.95 (425.05±4.70) µm×44.2-50.83 (47.88±2.85) µm. The anterior pole of R. porrecta and the present form possesses a very well developed mucorn which is made up of smooth hyaline protoplasm. But in R. pilosa this type of attaching apparatus surrounding the mucorn is lacking. The ectosarc of R. pilosa is thick, the cuticle thin with close longitudi- nal ridges. The cuticular hairs are also present in R. pilosa, but in R. porrecta the epicyte is thin, transparent and marked by 36 to 48 longitudinal ridges. Longitudinal myonems are also present in the myocyte of both R. pilosa and R. porrecta. Myonem is absent in the present form. The endoplasm of R. pilosa is viscous, packed with oval paraglycogen grains whereas the endoplasm of R. porrecta is composed of dense granular protoplasm within many ovoid paraglycogen grains which resemble the present species. Nucleus in the present form is small, elongated and found in the posterior portion of the gamont, measuring 17.68- 22.01 (19.74±1.95) µm×11.05-15.47 (13.40±1.95) µm. However, in R. pilosa the nucleus is rounded and large while it is of a vesicu- lar type in R. porrecta. The gametocysts of R. porrecta contain two approximately equal sized gametocytes. Spores are biconi- cal, symmetrical and measures 27.7 µm - 28.0 µm×11.8 to 12.1 µm in R. porrecta. The cyst of R. pilosa is ovoid and measures 94.9 µm×4.2 µm. They are comparatively small and measure 13.3 µm to 5.0 µm. The gametocysts of the present form are also ovoid with two unequal sized gametocytes and the gametocyst measures 95.03-97.24 (96.20±1.14) µm×77.35-81.77 (79.56±1.86) µm. Tye oocyst of the present form is biconical. It is 11.05-15.47 (12.81±1.71) µm in length and 6.63-8.84 (7.70±1.20) µm in width.
Besides these, the adult trophozoites of R. pilosa and R. porrecta undergo a process of asexual reproduction, plasmotomy. R. por- recta also undergo a process of degeneration in their life cycle.
However it is not found in the present species. Moreover, the hosts are also different. R. pilosa was described from the seminal vesicles of Lumbricus castanues, L. terrestris, Helodrilus foetidus while the R. porrecta was obtained from the seminal vesicles of L.
Table 1. Summarized statistics of different body parts of the gamonts, gametocysts and oocysts. All measurements are in µm
Characters Mean Range Standard
Deviation Gamont length (GL) 425.05 419.90-430.95 4.70 Gamont width at the 47.88 44.2-50.83 2.85 widest part (anterior part)
(GWwa)
Gamont width 16.50 15.47-17.68 1.14 (Posterior part) (GWp)
Length of nucleus (LN) 19.74 17.68-2.01 1.95 Width of nucleus (WN) 13.40 11.05-15.47 1.95 Length of gametocyst (LG) 96.20 95.03-97.24 1.14 Width of gametocyst (WG) .925 88.4-99.45 5.823 Large gametocyte 59.81 57.46-61.88 1.76 length (LGL)
Large gametocyte 72.48 70.72-75.14 1.71 width (LGW)
Small gametocyte 34.47 30.94-37.57 2.74 length (SGL)
Small gametocyte 64.38 61.88-66.3 1.84 width (SGW)
Length of oocyst (LO) 12.81 11.05-15.47 1.71 Width of Oocyst (WO) 7.70 6.63-8.84 1.25
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A New Gregarine Parasite Species
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rubellus and Helodrilus foetidus. On the contrary the present form has been described from the Metaphire peguana. A comparative table: Table 2 contains the comparison of the present species with other closely related species. It is seen from the table that the present form differs in morphology, morphometrics, host prefer- ence and distribution pattern from the other two previously described Rhynchocystis species. Moreover, it is evident that the present species obtained from Metaphire peguana is also totally different from the two closely related species R. porecta and R.
polisa. After careful consideration, it can be concluded that the present form should not be accommodated into any known spe- cies under the genus Rhynchocystis. Therefore, a separate status has been proposed for the present form and it is also described for the first time from Metaphire peguana of Bangladesh. Hence the name Rhynchocystis silvae sp. nov. is being proposed here.
Acknowledgments
One of the authors (SS) is grateful to the authorities of the University of Kalyani, Kalyani, West Bengal, India for extending permission to work at the Parasitology Laboratory of the Department of Zoology,
University of Kalyani, and Dr. C.K. Mandal of Zoological Survey of India, Calcutta for identification of the host specimen.
Conflict of Interest
No conflict of interest was declared by the authors.
REFERENCES
1. Ruston J. Dirhynchocystis minuta n. sp. gregarine from the seminal vesicles of Lumbricus terrestris L. with a note on association of Rhynchocystis porrecta. Schmidt J Parasit 1959; 45: 259-62. [CrossRef]
2. Bandyopadhyay PK, Mitra AK. Dirhynchocystis indica (Apicomplexa, Rhynchocystinae), a new gregarine species from the earthworm Lampito mauritii (Annelida, Oligochaeta) in India. Protistology 2006;
4: 207-11.
3. Levine ND. the protozoan phylum Apicomplexa. Vol.I.CRC Press Inc., Boca Raton, Florida USA, 1988; 37-8.
4. Clopton RE. Standard nomenclature and metrics of plane shapes for use in gregarine taxonomy. Comp Parasitol 2004; 71: 130-40. [CrossRef]
5. Troisi RA. Studies on the acephaline gregarines (Protozoa, Sporozoa) of some Oligochaete Annelids. The American Microscopical Society 1933; 62: 326-52. [CrossRef]
Species Characters R. pilosa Hesse (1909) R. porrecta Hesse (1909) R. silvae sp. nov Host(s) Lumbricus castaneus, Lumbricus rubellus and Metaphire peguana
Lumbricus terrestris and Helodrilus foetidus Helodrilus foetidus
Locality Europe (Philadelphia) Europe (Philadelphia) North Badda, Dhaka District,
(Bangladesh)
Gamonts Long and slender. Anterior Snake like with enlarged head. Elongated with a conical, end broad, rounded and Total length of the gamont is enlarged head and blunt posterior extremity is pointed. 21/2 mm. Anterior end swollen posterior ends. Mature gamont It measures 217.3µ in length by average 32µ to 36µ in diameter length 419.90-430.95
25.5µ in width and rest of the body tapers (425.05±4.70) µm and width at gradually to a point average the widest part is 44.2-50.83 28µ in diameter (47.88±2.85) µm. Width of the
posterior end is 15.47-17.68 (16.50±1.14) µm
Mucorn Present. “Cuticle hair” Present. But “Cuticle hair” Present at the anterior part.
is also present for attacking is absent It measures 13.26-17.68
to the host (15.47±1.86) µm×17.68-22.1
(19.44±1.71) µm
Longitudinal Myonems Present Present Absent
Nucleus Rounded, large with central Vesicular type Small, elongated and at the
karyosome and at the anterior posterior end. It measures
end 17.68-22.01 (19.74±1.95)
µm×11.05-15.47 (13.40±1.95) µm Endoplasm Viscous, packed with oval Composed of dense granular Composed of dense granular
paraglycogen grains protoplasm within many ovoid protoplasm with ovoid paraglycogen grains paraglycogen grains
Gametocysts Ovoid, and measures Speherical, contains two equal Ovoid, with two unequal sized 94.9µ×84.2µ sized gametocytes. Each gametocytes. Gametocysts
gametocyte produces measure 95.03-97.24 (96.20±1.14) isogametes µm×77.35-81.77 (79.56±1.86) µm Spores Small and measures 13.3µ to 5.0µ Biconical, symmetrical and Biconical and measures
measures 27.7µ to 11.05-15.47 (12.81±1.71) 28.0µ×11.8µ to 12.1µ µm×6.63-8.84 (7.70±1.20) µm
Plasmotomy Present Present Absent
Degeneration Absent Present Absent
References Troisi, 1933 Troisi, 1933 Present study
Table 2. A comparison between different characters of R. silvae sp. nov. with closely related species is presented. All measurements are in µm
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A New Gregarine Parasite Species
